Epitope and its use of hepatitis b virus surface antigen

ABSTRACT

Disclosed are an epitope specific to hepatitis B virus (HBV) and use thereof. The disclosed epitope is a conservative position on which mutagenesis does not occur and, therefore, a composition including an antibody to the foregoing epitope or a vaccine composition including the epitope has very low possibility of causing degradation of curing efficacy due to HBV mutation, thus being very useful for HBV treatment.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. application Ser.No. 14/127,052 (allowed) filed Dec. 17, 2013 which is a National Stageof International Application No. PCT/KR2011/005477 filed Jul. 25, 2011,claiming priority based on Korean Patent Application No. 10-2011-0064671filed Jun. 30, 2011, the contents of all of which are incorporatedherein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to an epitope specific to Hepatitis Bvirus (hereinafter, referred to as ‘HBV’) and use thereof. Since theepitope disclosed herein is a conservative position on whichmodification due to mutation (‘mutagenesis’) does not occur, acomposition including an antibody against the epitope or a vaccinecomposition including the epitope described above has very lowpossibility of causing degradation of curing efficacy by HBV mutation,thus being very useful for HBV treatment.

The present invention also relates to a method for production of anantigen specific antibody to the epitope described above and suchantigen specific antibody to the epitope produced according to thepresent invention exhibits excellent specificity when administered invivo.

BACKGROUND ART

HBV is a virus having DNA genomes belonging to Hepadnaviridae family andcauses acute and/or chronic hepatitis. In general, HBV is classifiedinto eight genotypes which have at least 8% different gene sequences toone another or, otherwise, divided into nine serotypes (i.e., adw, adr,ayw, ayr, or the like) on the basis of two antigenic determinants (thatis, epitopes) (d/y, w/r) of HBV surface antigen (HBsAg). 350 millionpeople worldwide have been infected with chronic HBV and, specifically,about 5 to 8% of the population in Korea and China has chronic HBVinfection. HBV infection is a major cause of liver diseases and livercancer in these regions. At present, although the above infection can beprotected somewhat by the development of vaccines, lots of patientsstill suffer from chronic Hepatitis B infection caused by HBV.HBV-caused chronic infection may induce hepatitis as well as livercirrhosis and liver cancer and, as compared to non-infected people,people with chronic infection show an increase in liver cancer about 300times higher. According to WHO investigation, chronic hepatitis B isconsidered as a major cause of about 80% of liver cancers.

Chronic hepatitis B medicine recently developed as a nucleoside analogueand available on the market may include, for example, lamivudine,adefovir dipivoxil, etc. These medicines may interfere with a reversetranscriptase of HBV polymerase, in turn inhibiting HBV DNA replication.However, in the case where any one of the foregoing medicines isadministered for a long term such as 3 years, about 75% of the patientshave drug resistance viruses, thus entailing a problem of deteriorationin the curing efficacy. In order to prevent vertical transmission orinfection after liver transplantation, the foregoing medicines arecommonly used with hepatitis B immunoglobulin (HBIG).

Currently HBIG is manufactured by ion-exchange purification and virusinactivation from plasma of donors with high anti-HBsAg antibody titer.

However, the currently available HBIG is not an ideal source oftherapeutic antibody due to its limited availability, low specificactivity and possible contamination of infectious agents.

It is known that antibodies generated in vivo by vaccines now used inthe art are mostly antibodies recognizing ‘a’ epitope of HBV. However,mutants escaping such antibodies, for example, a G145R mutant generatedby substituting glycine at 145 of the HBsAg with arginine has recentlybeen reported. Additionally, a variety of escaping mutants have alsobeen found, therefore, existing HBV medicines involve limitations inrendering satisfactory curing efficacy. Accordingly, there is anincreasing demand for HBV treatment antibodies and/or HBV vaccinesspecifically bound to epitopes that correspond to sites necessary forthe survival of HBV in association with HBV replication and does notcause mutation, thus not causing deterioration in curing efficacy due tomutation.

DISCLOSURE Technical Problem

In order to solve the problems described above, the present inventionprovides HBV specific epitopes including RFLWE (SEQ ID NO: 4) or KFLWE(SEQ ID NO: 5) and, in particular, an epitope having an amino acidsequence such as FARFLWEWASVRFSW (SEQ ID NO: 6) or FGKFLWEWASARFSW (SEQID NO: 7) that is a necessary site for the survival of HBV, thuscorresponding to a conservative position on which mutation does notoccur.

Another object of the present invention is to provide methods forproduction of the epitope described above, a HBV vaccine composition orvaccine comprising the epitope and an antibody capable of specificallybinding to the epitope by applying the foregoing epitope, as well as aHBV treatment composition or curing agent including the antibodyproduced as described above.

A still further object of the present invention is to provide acomposition or kit for HBV detection having the epitope described aboveor a polynucleotide sequence encoding the epitope.

Technical Solution

The inventors of the present invention have found that; epitopes of ahuman antibody specifically binding to a HBV surface antigen (seePCT/KR2010/004445, hereinafter referred to as the ‘inventive antibody’)correspond to sequences including RFLWE (SEQ ID NO: 4) or KFLWE (SEQ IDNO: 5) and, in particular, sequences derived from FARFLWEWASVRFSE (SEQID NO: 6) or FGKFLWEWASARFSE (SEQ ID NO: 7) or a part thereof; and suchepitope sites are favorably conservative, significant for HBVreplication and necessary for HBV survival. Therefore, the presentinvention has been completed under the foregoing discovery. Among theafore-mentioned epitopes, the epitopes having SEQ ID NO. 4 and SEQ IDNO. 6 are epitopes of adr subtypes (SEQ ID NO: 1) of HBV while theepitopes having SEQ ID NO. 5 and SEQ ID NO. 7 correspond to epitopes ofayw subtypes (SEQ ID NO: 2) of HBV.

The HBV specific epitope defined by any one of SEQ ID NOS. 4 to 7according to the present invention may retain a three-dimensionalstructure or may be used as a conjugated form with a carrier, in orderto improve efficiency when used for a composition such as a vaccine. Thecarrier used herein may include any one, which is bio-available andrenders desired effects of the present invention, and be selected frompeptide, serum albumin, immunoglobulin, hemocyanin, polysaccharides, orthe like, without being particularly limited thereto.

The HBV specific epitope defined by any one of SEQ ID NOS. 4 to 7 assuch or a composite thereof combined with a carrier may be useable as avaccine composition for HBV treatment. In this regard, the vaccinecomposition may further include a pharmaceutically acceptable adjuvantor excipient. Such an adjuvant serves to facilitate formation of anantibody by injecting in vivo the adjuvant, and may include any oneenabling achievement of purposes of the present invention, moreparticularly, at least one selected from aluminum salts (Al(OH)₃,ALPO₄), squalene, sorbitane, polysorbate 80, CpG, liposome, cholesterol,monophosphoryl lipid (MPL) A and glucopyranosyl lipid (GLA) A, withoutbeing particularly limited thereto.

A polynucleotide encoding the HBV specific epitope defined by SEQ IDNOS. 4 to 7 and provided according to the present invention may be usedas DNA vaccine. Here, the polynucleotide may be used as such without anyvector or, otherwise, supported in a viral or non-viral vector. Theviral or non-viral vector used herein may include any one commonlyavailable in the art (to which the present invention pertains). Theviral vector preferably includes adenovirus, adeno-associated virus,lentivirus, letrovirus, etc., while the non-viral vector may include acationic polymer, a non-ionic polymer, liposome, lipid, phospholipid, ahydrophilic polymer, a hydrophobic polymer and a combination of at leastone selected from the foregoing materials, without being particularlylimited thereto.

The present invention provides a recombinant vector including apolynucleotide that encodes the HBV specific epitope defined by any oneof SEQ ID NOS. 4 to 7 according to the present invention, a host cellincluding the recombinant vector, and a method for production of the HBVspecific epitope defined by any one of SEQ ID NOS. 4 to 7 according tothe present invention, using the recombinant vector or host celldescribed above.

In the present invention, the ‘recombinant vector’ is an expressionvector that represents a target protein from a suitable host cell whichis a gene product containing a necessary regulating element operablylinked to a gene insert to express the gene insert. In the presentinvention, the term ‘operably linked’ refers to a nucleic acidexpression control sequence functionally linked to a nucleic acidsequence encoding the target protein, so as to execute generalfunctions. The operable linkage with the recombinant vector may beperformed by gene recombination technologies well known in the art towhich the present invention pertains. Site-specific DNA cleavage andlinkage may also be easily performed using enzymes commonly known in theart to which the present invention pertains.

Appropriate expression vectors useable in the present invention mayinclude signal sequences for membrane targeting or secretion as well asexpression control elements such as a promoter, a start codon, a stopcodon, a polyadenylated signal, an enhancer, or the like. The startcodon and stop codon are generally considered as a part of a nucleotidesequence encoding an immunogenic target protein and, when administeringa gene product, must exhibit an action in an individual while beingin-frame with a coding sequence. The general promoter may be structuralor inductive. A prokaryotic cell may include, for example, lac, tac, T3and T7 promoters, without being particularly limited thereto. Aneukaryotic cell may include, for example, monkey virus 40 (SV40), amouse breast tumor virus (MMTV) promoter, human immunity deficient virus(HIV) and, in particular, a long terminal repeat (LTR) promoter of HIV,Moloney virus, cytomegalovirus (CMV), Epstein bar virus (EBV), Roussarcoma virus (RSV) promoter, as well as β-actin promoter, humanhemoglobin, human muscle creatin, human metallothionein derivedpromoter, without being particularly limited thereto.

The expression vector may include a selection marker to select a hostcell containing a vector. The selection marker functions to sort cellstransformed into vectors and may include markers providing selectablephenotypes such as drug resistance, nutrient requirements, tolerance tocellular cytotoxicity, expression of surface protein, etc. Since cellsexpressing the selection marker under selective agent-treated conditionsonly are alive, transformed cells may be screened. For a replicableexpression vector, the vector may have a replication origin as aparticular nucleic acid sequence at which replication starts. Theexpressed recombinant vector may include a variety of vectors such asplasmid, virus, cosmid, etc. The recombinant vector is not particularlylimited so long as various host cells of prokaryotes and eukaryotesexpress desired genes and produce desired proteins, however, ispreferably a vector to produce a great quantity of foreign proteinssimilar to a natural one, which possess a promoter having strongactivity while attaining strong expression.

In particular, in order to express HBV specific epitopes defined by anyone of SEQ ID NOS. 4 to 7, a variety of expression host-vectorcombinations may be used. An expression vector suitable for eukaryotemay include expression control sequences derived from; for example,SV40, bovine papilloma virus, adenovirus, adeno-associated virus,cytomegalovirus, lenti-virus and/or retro-virus, without beingparticularly limited thereto. The expression vector used for bacteriahosts may include, for example: bacterial plasmids obtained fromEscherichia coli such as pET, pRSET, pBluescript, pGEX2T, pUC vector,col E1, pCR1, pBR322, pMB9, and derivatives thereof; plasmids such asRP4 with a wide range of hosts; phage DNA exemplified as various phagelambda derivatives such as λgt10 and λgt11, NM980, etc.; other DNAphages such as single-stranded filament type DNA phage, M13, or thelike. A vector useful for insect cells may be pVL941.

The recombinant vector is inserted in a host cell to form a transformantand the host cell suitably used herein may include, for example:prokaryotes such as E. coli, Bacillus subtilis, Streptomyces sp.,Pseudomonas sp., Proteus mirabilis or Staphylococcus sp.; fungi such asAspergillus sp.; yeasts such as Pichia pastoris, Saccharomycescerevisiae, Schizosaccharomyces sp., Neurospora crassa, etc.; eukaryoticcells such as lower eukaryotic cells, higher eukaryotic cells, i.e.,insect cells, or the like. The host cell is preferably derived fromplants and/or mammals and, in particular, derived from monkey kidneycells 7 (COST), NSO cells, SP2/0, Chinese hamster ovary (CHO) cells,W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78cells and/or HEK293 cells, without being particularly limited thereto.Most preferably, CHO cells are used.

In the present invention, the term ‘transformation into host cells’includes any technique for introduction of nucleic acid into organics,cells, tissues and/or organs and, as well known in the conventional art,a standard technique may be suitably selected depending upon the hostcells to perform the transformation. Among such techniques,electroporation, protoplasm fusion, calcium phosphate (CaPO₄)precipitation, calcium chloride (CaCl₂) precipitation, agitation usingsilicon carbide fibers, agro-bacteria mediated transformation,transformation mediated with PEG, dextrane sulfate and lipofectamine andthrough drying/inhibition, without being particularly limited thereto.By incubating a transformant in which the recombinant vector isexpressed in a culture medium, the HBV specific epitope defined by anyone of SEQ ID NOS. 4 to 7 may be formed in large quantities. The culturemedium and culturing conditions may be suitably selected among thosecommonly used depending on host cells being used. During culturing, someconditions such as a temperature, pH of the medium, a culturing time,etc., may be controlled to enable appropriate cell growth andmass-production of proteins. As described above, the HBV specificepitope defined by any one of SEQ ID NOS. 4 to 7 may be collected fromthe medium or cell decomposition product by a recombination way andseparated or purified by any conventional biochemical separationtechnique (Sambrook et al., Molecular Cloning: A Laboratory Manual,2^(nd) Ed., Cold Spring Harbor Laboratory Press (1989); Deuscher, M.,Guide to Protein Purification Methods Enzymology, Vol. 182. AcademicPress, Inc., San Diego, Calif. (1990)). For this purpose, variousmethods such as electrophoresis, centrifugation, gel filtration,precipitation, dialysis, chromatography (ion-exchange chromatography,affinity chromatography, immune-adsorption chromatography, sizeexclusion chromatography, etc.), isoelectric point focusing, and variousvariations and combinations thereof may be utilized, without beingparticularly limited thereto.

The present invention provides a method for expressing the HBV specificepitope defined by any one of SEQ ID NOS. 4 to 7 on the surface ofmicroorganisms or virus. In this case, a recombinant vector including asequence that encodes an inducing promoter or a signal protein, as wellas various microorganisms or viruses having the above recombinant vectormay be used. More particularly, recombinant E. coli, yeast and/orbacteriophage are appropriate microorganisms and/or viruses, withoutbeing particularly limited thereto. In order to express the HBV specificepitope defined by any one of SEQ ID NOS. 4 to 7 on the surface of theforegoing microorganisms or viruses, display techniques well known inthe art to which the present invention pertains may be used.Specifically, a polynucleotide sequence encoding the HBV specificepitope defined by any one of SEQ ID NOS. 4 to 7 may be combined with(or bound to) a sequence encoding a promoter or a signal protein thatderives expression on the surface of a microorganism cell or virus, thusexpressing the HBV specific epitope. Alternatively, after deleting apart of gene sites at which the surface expressing protein is encoded, apolynucleotide sequence encoding the HBV specific epitope defined by anyone of SEQ ID NOS. 4 to 7 may be inserted into the deleted part.However, the present invention is not particularly limited to theforegoing methods. According to the afore-mentioned methods, the HBVspecific epitope defined by any one of SEQ ID NOS. 4 to 7, which isexpressed on the surface of the microorganism or virus, may be separatedas such and purified for desired uses according to the presentinvention. In addition, the inventive epitope may be used to screen anantibody specifically bound to the HBV specific epitope defined by anyone of SEQ ID NOS. 4 to 7, which is expressed on the surface, and thenobtaining the screened antibody.

Furthermore, the present invention provides a method for production ofan antibody specific bound to the HBV specific epitope defined by anyone of SEQ ID NOS. 4 to 7, or fragments of the antibody, which includesusing the HBV specific epitope defined by any one of SEQ ID NOS. 4 to 7,a composite containing the foregoing epitope or a polynucleotideencoding the foregoing epitope. Such antibody may be a polyclonalantibody or monoclonal antibody and, so long as fragments thereof havecharacteristics of being bound to the HBV specific epitope defined byany one of SEQ ID NOS. 4 to 7, they are also included within the scopeof the present invention. More particularly, the inventive antibody orfragments thereof may include, for example: single-chain antibodies;diabodies; triabodies; tetrabodies; Fab fragments; F(ab′)₂ fragments;Fd; scFv; domain antibodies; dual-specific antibodies; minibodies; scap;IgD antibodies; IgE antibodies; IgM antibodies; IgG1 antibodies; IgG2antibodies; IgG3 antibodies; IgG4 antibodies; derivatives inantibody-unvariable regions; and synthetic antibodies based on proteinscaffolds, all of which have the binding ability to the HBV specificepitope defined by any one of SEQ ID NOS. 4 to 7, without beingparticularly limited thereto. So long as characteristics of theinventive antibody are retained, antibodies mutated in variable regionsmay also be included within the scope of the present invention. This maybe exemplified by conservative substitution of an amino acid in avariable region. Here, such ‘conservative substitution’ usually refersto substitution of an amino acid into another amino acid residue havingsimilar properties to the original amino acid sequence. For example,lysine, arginine and histidine have base side-chains, in turn showingsimilar properties. On the other hand, both aspartic acid and glutamicacid have acid side-chains and exhibit similar properties to each other.In addition, glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine and tryptophan are similar to one another since theyhave non-charged polar side-chains, while alanine, valine, leucine,threonine, isoleucine, proline, phenylalanine and methionine are similarto one another since they have non-polar side-chains. Further, tyrosine,phenylalanine, tryptophan and histidine are similar to one another sincethey have aromatic side-chains. Consequently, it will be obvious tothose skilled in the art that, even though amino acid substitutionoccurs within any one of the foregoing groups having similar properties,significant change in characteristics may not be found. Therefore, ifspecific properties of the inventive antibody are retained, a method forproduction of antibodies having mutated due to conservative substitutionin a variable region may also be included within the scope of thepresent invention.

The antibody bound to the HBV specific epitope defined by any one of SEQID NOS. 4 to 7 may be prepared by any conventional method known in theart (to which the present invention pertains). More particularly, afterinoculating an animal with the HBV specific epitope defined by any oneof SEQ ID NOS. 4 to 7, a composite including the epitope or apolynucleotide encoding the epitope described above, an antibodyspecifically bound to the HBV specific epitope defined by any one of SEQID NOS. 4 to 7 is produced and screened from the inoculated animal, inturn being obtainable.

The animal used herein may include a transgenic animal, in particular, atransgenic mouse capable of producing the same antibody as ahuman-derived sequence. The so-called fully human antibody havingdecreased immunogenicity, which is obtained using a transgenic mouse,may be produced according to any one of the methods disclosed in: U.S.Pat. Nos. 5,569,825; 5,633,425; and 7,501,552, or the like. In the casewhere the afore-mentioned animal has not been preferably transformed toallow production of the same antibody as the human-derived sequence, ahumanization or deimmunization process may be further implemented, usingthe antibody obtained from the animal, according to any one of themethods disclosed in: U.S. Pat. Nos. 5,225,539; 5,859,205; 6,632,927;5,693,762; 6,054,297; 6,407,213; and WO Laid-Open Patent No. 1998/52976,thus suitably processing the antibody to be useful for in vivotreatment. More particularly, such humanization or deimmunization mayinclude CDR-grafting to graft a CDR sequence of an antibody producedfrom an animal into a framework of a human antibody and, in order toincrease affinity or decrease immunogenicity, further include aCDR-walking process to substitute, insert and delete at least one aminoacid sequence.

Instead of the HBV specific epitope defined by any one of SEQ ID NOS. 4to 7, a composite including the epitope and/or a polynucleotide encodingthe epitope, if the overall HBV is used as an immunogen, a process ofpredominantly screening (often ‘panning’) antibodies having HBV bindingability (sometimes abbreviated to ‘binding’) and then additionallypanning antibodies to specifically recognize the HBV specific epitopedefined by any one of SEQ ID NOS. 4 to 7, among the primarily screenedantibodies, may be used. Alternatively, a method for screeningantibodies, which have no binding or decreased binding to HBVs mutatedat important sites of the HBV specific epitope defined by any one of SEQID NOS. 4 to 7, among primarily screened HBV binding antibodies, whereinthe method includes deriving mutation at the important sites of the HBVspecific epitope defined by any one of SEQ ID NOS. 4 to 7, may also beused.

Meanwhile, according to display techniques well known in the art, humanantibodies bound to the HBV specific epitope defined by any one of SEQID No. 4 to 7 may be produced and screened. Such display techniques maybe selected from a phage display, a bacterial display or a ribosomedisplay, without being particularly limited thereto. Production anddisplay of libraries may be easily performed according to theconventional art disclosed in, for example; U.S. Pat. Nos. 5,733,743,7,063,943, 6,172,197, 6,348,315, 6,589,741, or the like. Especially, thelibraries used in the foregoing display may be designed to have thesequences of human-derived antibodies. More particularly, the methoddescribed above may be characterized by screening (or panning)antibodies specifically bound to the HBV specific epitope defined by anyone of SEQ ID NOS. 4 to 7 only, by applying the HBV epitope defined byany one of SEQ ID NOS. 4 to 7 or a composite including the epitope.

Finally, the present invention provides a HBV detecting composition orkit, which includes the epitope defined by any one of SEQ ID NOS. 4 to7, a composite including the epitope or a polynucleotide encoding theepitope. The HBV detecting composition or kit according to the presentinvention may have merits of enabling rapid and precise diagnosis of HBVinfection while not under significant influence of HBV mutation. The HBVdetection kit, which includes the epitope defined by any one of SEQ IDNOS. 4 to 7, a composite including the epitope or a polynucleotideencoding the epitope, may be fabricated to utilize a variety of methodsincluding, for example, a general enzyme-linked immunosorbent assay(ELISA), a fluorescence-activated cell sorting (FACS) method, or thelike. Moreover, in the case where the polynucleotide encoding theepitope of the present invention is used, hybridization may be detectedby common hybridization techniques

Advantageous Effects

As is apparent from the detailed description, the HBV specific epitopeprovided according to the present invention is substantially aconservative position on which mutagenesis does not occur. Therefore, acomposition or vaccine composition including an antibody against theforegoing epitope has relatively low possibility of causingdeterioration in curing efficacy by such HBV mutation, thereby beingeffectively used in HBV treatment and/or diagnosis.

DESCRIPTION OF DRAWINGS

The above and other objects, features and advantages of the presentinvention will become apparent from the following description ofpreferred embodiments given in conjunction with the accompanyingdrawings, in which:

FIG. 1 illustrates analysis results of variation in binding ability toHBV surface antigen protein mutants in order to identify epitopes of theinventive antibody;

FIG. 2 shows a loop structure in HBV surface antigen protein includingthe inventive epitope;

FIG. 3 illustrates a HBV genomic structure wherein the genome S ORFencoding the surface antigen protein is partially overlapped with thegenome P ORF encoding a polymerase;

FIG. 4 illustrates a process of preparing mutants of the HBV polymerase;

FIG. 5 illustrates a complementation test process executed by infectingHepG2 cell with a HBV Pol-free replicon and a HBV polymerase mutant,simultaneously;

FIG. 6 shows test results of HBV replication ability of each HBVpolymerase mutant through Southern blot analysis (comparison of HBV DNAreplication intermediates, i.e., RC, DL, SS DNA at the right side of thegraph);

FIG. 7 shows test results of influences upon pregenomic RNA packaging byrespective HBV polymerase mutants through RNase protection assay; and

FIG. 8 shows a linkage map of HBV gene vector used in hydrodynamicinjection in order to generate HBV virus particles in a mouse.

BEST MODE

Hereinafter, preferred embodiments of the present invention will bedescribed in detail with reference to examples, however, such examplesare for illustrative purposes only and not intended to limit the scopeof the present invention.

Example 1 Identification of Epitope of Inventive Antibody

In order to identify the epitope of the inventive antibody, aftercausing random mutagenesis in the surface antigen protein of HBV adrsubtypes (see SEQ ID NO. 1), binding of the inventive antibody torespective mutants was investigated. Here, preparation of the mutantsand assay of the binding of the inventive antibody were implementedaccording to shotgun mutagenesis available from Integral Molecular Co.(J Am Chem Soc. 2009; 131(20): 69526954). Characteristics of mutationlibraries used for identifying the epitope are shown in the followingTable 1. After infecting HEK-293T cells with clones having the abovelibraries, the binding of the inventive antibody was assayed byimmune-fluorescence assay.

The binding of the inventive antibody was determined by averagingresults from tests repeated three times and subjected to normalizationbased on the binding of a wild type HBV surface antigen protein. In thiscase, using a rabbit polyclonal antibody against the HBV surface antigenprotein, expression of the mutated surface antigen protein and thebinding of the inventive antibody to such expression were investigated.

TABLE 1 Characteristics of library used for epitope identificationNumber of clones in library 441 Amino acid residues (AAs) of mutated 223(of total HBV surface antigen 226) Average number of AA mutations per 1.2 clone Average number of mutations per AA  2.4 residue Number(percentage) of AAs mutated at 223 (99%) least once Number (percentage)of AAs mutated at 216 (96%) least twice Number (percentage) of clones357 (81%) containing a single AA mutation Number (percentage) of clones 76 (17%) containing two AA mutations Number (percentage) of clones  8(2%) containing more than two AA mutations

From the table, it was found that the inventive antibody lost thebinding ability to eight (8) clones having mutation occurring at threeamino acid residues (AAs) of the HBV surface antigen protein (see FIG.1). That is, for the eight clones shown in FIG. 1, it was confirmed thatthe rabbit polyclonal antibody exhibited the binding ability, in turnnormally expressing the mutated HBV surface antigen protein, however,the inventive antibody was not bound thereto.

As a result of assaying the eight clones, it was found that each has atleast one mutation at 160R (160R means the amino acid R located atposition 160, hereinafter the same as above), 163W and 164E (SEQ ID NO.1), respectively. That is, the above sequence may be determined as asite corresponding to the epitope of the inventive antibody. From suchresult, it was found that the epitope of the inventive antibody containsRFLWE (SEQ ID NO. 4) and the epitope in ayw subtype of HBV with thebinding ability contains KFLWE (SEQ ID NO. 5).

Specifically, the epitope having the sequence defined by SEQ ID NOS. 4or 5 may be FARFLWEWASVRFSW (SEQ ID NO. 6) or FGKFLWEWASARFSW (SEQ IDNO. 7) corresponding to a minor loop among two loops at HBV surface siteat which the above epitope is present (see FIG. 2).

Example 2 Identification of Characteristics of Epitope of InventiveAntibody

(1) Preparation of HBV Polymerase (HBV Pol) Mutants

Epitopes of the inventive antibody include 160K, 163W and 164E (SEQ IDNO. 2) in the surface antigen ORF (S ORF) of the HBV ayw subtype,wherein the ORF sequence of the HBV surface antigen encoding theepitopes overlaps with HBV P ORF encoding the HBV polymerase. Inparticular, 5041, 506M, 507G and 508V (see SEQ ID NO. 3) of the HBVpolymerase may correspond to the sites at which the epitope is encodedby genes in the OFR encoding the epitope (see FIG. 3). Briefly, mutationat the foregoing sites in the HBV S ORF also involves mutation of theHBV P ORF.

The HBV polymerase has remarkably different features from other viralpolymerases. First, the HBV polymerase has reverse transcriptaseactivity that synthesizes it's DNA from RNA (pregenomic RNA: pgRNA);second, during reverse transcription initiation, the HBV polymerase usesitself as the primer to conduct protein-priming; and third, primertranslocation and template switching are executed during replication,although the correct mechanism is not still identified.

Meanwhile, as described above, an open reading frame (‘ORF’) thatencodes the epitope site of the inventive antibody neutralizing HBV,that is, the epitope site of the inventive antibody in the HBV surfaceantigen, may overlap with another ORF encoding the HBV polymerase.Therefore, in order to survey influence by the HBV polymerase site,which is encoded by the HBV P ORF overlapping with the ORF encoding theepitope of the inventive antibody, upon HBV virus replication, mutationpossibility of the foregoing epitope was investigated.

For this purpose, a mutant substituting an amino acid, which is presentat the site overlapping with the epitope of the inventive antibody inthe HBV P ORF, into an alanine, was prepared through manipulation andsubjected to survey of influence of the prepared mutant upon reversetranscriptase activity of a HBV polymerase (‘HBV Pol’). First, themutants such as K503A (K503A means that the amino acid K at the site 503is mutated into A, hereinafter the same as above) 1504A, M506A, G507Aand V508A, which are obtained by substituting 503K, 5041, 506M, 507G and508V of the HBV Pol polymerase with alanines, as well as a naturallygenerated mutant V508L have been prepared as shown in FIG. 4. Then, thevariation in genome replicating function of the HBV polymerase having amutant at the foregoing epitope site, has been investigated throughcomplementation tests. In particular, HBV Pol-null replicon as a HBVmutant in which frame-shift mutation is derived in HBV P ORF and towhich the HBV polymerase shows lack of activity, as well as a plasmidexpressing the HBV polymerase in which mutation is derived as describedabove, have been infected HepG2 cells (see FIG. 5). Thereafter, HBVgenome replication was assayed by Southern blot analysis and RNaseprotection assay (RPA).

(2) Southern Blot Analysis

As described above, the HBV Pol-null replicon and the mutant derivingmutation of the HBV polymerase have simultaneously infected HepG2 cell,followed by collection of replicated virus DNAs after 4 days. Thecollected materials were subjected to assessment of HBV DNA replication.

As a result, for K503A mutant, virus DNA replication was about 17%,compared to wild type. This result indicates that 503K site in the HBVpolymerase significantly participates in a mechanism of virus DNAreplication. On the contrary, M506A and G507A mutants have rarely showedvirus DNA replication. This fact demonstrates that 506M and 507G areessential sites for virus DNA replication mechanism of the HBVpolymerase. 1504A, V508A and V508L mutants exhibited respectively about65%, 70% and 82% of virus DNA replication, compared to the wild type.That is, it was observed that these mutants have received virus DNAreplication substantially similar to that of the wild type.Consequently, it was determined that the above mutants have relativelylow participation in HBV DNA replication (see FIG. 6).

(3) Results of RPA (RNase Protection Assay)

As a pre-stage before DNA replication, encapsidation of RNA (pregenomicRNA: pgRNA) was assayed via a RPA method (see Kim et al., 2009, J.Virol. 83: 8032-8040).

As described above, the HBV Pol-null replicon and the mutant derivingmutation of the HBV polymerase have simultaneously infected HepG2 cell,followed by collection of cores of the virus and total pgRNAs in cellsafter 3 days. The collected materials were subjected to quantitativeassay of pgRNA packaging extent wherein the pgRNA is used as a templatefor HBV DNA replication.

From the results, K503A and G507A mutants showed about 25% pgRNApackaging, compared to the wild type. This indicates that 503K and 507Gsignificantly participate in packaging of the pgRNA into core particlesof the virus. On the other hand, M506A mutant exhibited about 71% pgRNApackaging, compared to the wild type. That is, it was found thatparticipation of 506M to pgRNA packaging is relatively low. Othermutants, i.e., 1504A, V508A and V508L mutants showed pgRNA packagingsubstantially equal to the wild type, therefore, it is considered thatthese sites participate very little in pgRNA packaging (see FIG. 7).

(4) Overall Review for Influence of HBV Polymerase Mutants Upon HBVReplication

For K503A mutant of the HBV polymerase, only 25% pgRNA packagingresulted, compared to the wild type. As a result of quantifying thevirus DNA as a final product of the virus replication, it was found thatthe replication was accomplished only to the extent of the pgRNApackaging. Accordingly, it is deemed that the 503K site mostlyparticipates in the initial pgRNA packaging (see TABLE 2). On the otherhand, M506A mutant of the HBA polymerase exhibited about 71% pgRNApackaging, which is substantially similar to that of the wild type.However, quantification results of virus DNAs as a final product of thevirus replication revealed no replication. This fact means that,although M506 of the HBV polymerase never participates in pgRNApackaging, the M506 may significantly participate in a mechanism ofvirus DNA replication to synthesize (−)-strand DNAs using pgRNA as atemplate, i.e., a reverse transcription mechanism such as proteinpriming or primer translocation.

For G507A mutants of the HBV polymerase, pgRNA packaging was only 24% ofthe wild type and the virus DNA replication was executed very littleand, therefore, it may be considered that M507 site has importantfunctions in both the pgRNA binding and the reverse transcription of thepolymerase. Further, the M507 site may have a role in interaction with aprotein such as Hsp90 as a host factor and/or a core protein of the HBV,during encapsidation.

Meanwhile, the remaining mutants 1504A, V508A and V508L of the HBVpolymerase show pgRNA packaging and/or virus DNA replicationsubstantially similar to those of the wild type. Accordingly, amongsequences of the HBV polymerase that is encoded by HBV P ORF overlappingwith HBV S ORF which encodes HBV surface antigen protein sites 160K,163W and 164E found as the epitope of the inventive antibody, 160K and163W sites are in close association with the virus replication. In thecase where mutation is derived at these sites, virus replication may notbe executed, thus being high conservative positions. Accordingly, theabove two mutants do not exist and a specific-bound antibody to theforegoing sites may be effective in treating naturally generated mutantsand/or mutants exhibiting tolerance by anti-viral medicines.

TABLE 2 Replication ability and RNA packaging characteristics of HBVpolymerase mutants Mutant RNA packaging* DNA replication* HBV K503A + +polymerase I504A +++ ++ M506A ++ − G607A + − V509A +++ ++ V508L +++ +++*Compared to the wild type, +++: 70 to 100%; ++: 30 to 70%; +: 10 to30%; and −: <1%

Example 3 Binding and Neutralization Effects of Inventive Antibody toEpitope Mutants

(1) Preparation of Mutants

At least one of 163W and 164E (SEQ ID NO. 1) of the HBV surface antigenprotein (HBsAg), which are epitopes of the inventive antibody, wassubstituted by alanine, preparing a mutant. Since 160K relevant toserotypes has a problem in mutation, mutants thereof were excluded. Inaddition, mutants obtained by mutation of 164E into 164D have recentlybeen reported, therefore, mutants of E164D were also prepared and used.Since the mutants were obtained as described above, mutation was alsoderived at 506M, 507G and 508V (SEQ ID NO. 2) of the HBV polymeraseencoded by HBV P ORF overlapping with HBV S ORF which encodes theforegoing mutants. Here, even when the same amino acid mutation occursdepending upon variant codons at 163W and 164E of the HBV surfaceantigen protein, mutants of the HBV polymerase have different amino acidsequences (see TABLE 3).

TABLE 3 Mutants of HBsAg and Mutation of Corresponding HBV PolymeraseMutation HBsAg of HBV mutation polymerase Mutant before after beforeAfter M5-1 WE AA MGV SRL M5-2 AA SRV M5-3 AA SGL M5-4 AA SGV M5-5 AE SRVM5-6 AE SGV M5-7 WA MGL M5-8 WA MGM M5-9 WA MGV M6-1 WD MGL

(2) Test and Validation of In Vivo Efficacy Using Acute Hepatitis BDerived Mouse

By injecting HBV DNA into a C57BL6 mouse through hydrodynamic injectionto derive symptoms similar to acute hepatitis B, the treated mouse wasused to investigate binding of the inventive antibody, binding of HBVand/or HBV neutralization ability in the blood of the mouse whereepitope mutation was derived as described above. The used C57BL6 mousewas a 6-week aged female with about a weight of 20 g, which is purchasedfrom Charles Liver Laboratory (the United States). As shown in TABLE 4,a total of 12 groups with five mice per group were tested.

TABLE 4 Test conditions using C57BL6 mouse Number of Test material andSubject Individuals administering route Dose Wild type HBV 5 PBS, IV 0.2mL Wild type HBV 5 0.1 mg of inventive 0.2 mL antibody, IV M5-1 5 0.1 mgof inventive 0.2 mL antibody, IV M5-2 5 0.1 mg of inventive 0.2 mLantibody, IV M5-3 5 0.1 mg of inventive 0.2 mL antibody, IV M5-4 5 0.1mg of inventive 0.2 mL antibody, IV M5-5 5 0.1 mg of inventive 0.2 mLantibody, IV M5-6 5 0.1 mg of inventive 0.2 mL antibody, IV M5-7 5 0.1mg of inventive 0.2 mL antibody, IV M5-8 5 0.1 mg of inventive 0.2 mLantibody, IV M5-9 5 0.1 mg of inventive 0.2 mL antibody, IV M6-1 5 0.1mg of inventive 0.2 mL antibody, IV

Each mouse was treated by injecting 20 μg of pHBV-MBRI vector (Shin etal., Virus Research 119, 146-153, 2006; see FIG. 8) that contains HBVDNA sequence inserted in pcDNA3.1 (Invitrogen, the United States)through a tail vein of the mouse at 0.3 mL/min with a ratio of 9.5% byvolume per weight of the mouse, thus causing acute hepatitis B. Afterhours, as shown in TABLE 4, 0.2 mL of the inventive antibody wasintravenously (IV) administered through the tail vein of the mouse.Before injection of the inventive antibody (24 hours, 48 hours) andafter injection thereof (72 hours, 96 hours), the serum was separatedand diluted to 10 times in a goat serum, followed by measuring aconcentration in the blood of the HBV surface antigen protein (HBsAg)through Genedia HBsAg ELISA 3.0 (Green Cross Corp. MS, Korea). Withregard to HBV DNA, before (48 hours) and after (72 hours) the injectionof the inventive antibody, the blood was separated and analyzed by realtime PCR to perform quantitative assay of HBV DNA in blood, and then,comparative assay of HBV neutralization ability of the inventiveantibody.

As a result of detecting HBsAg in blood via Genedia HBsAg ELISA 3.0, itwas confirmed that, if 10 mutants are inserted, all HBsAgs are suitablyexpressed. When 10 variant type HBsAgs were assayed on binding to theinventive antibody, the variant HBsAg in which both 163W and 164E weresubstituted with alanine, did not show binding to the inventiveantibody. On the other hand, it was found that the variant HBsAg inwhich 163W only was substituted with alanine, shows the binding abilityof 70% or higher, compared to the wild type. In addition, the variantHBsAg having 164E substituted with alanine exhibited the binding abilityof about 30%, compared to the wild type. For E164D variant, bindingcharacteristics were substantially similar to the wild type (see TABLE5).

Mutation in HBsAg influences the sequences of the HBV polymerase asdescribed above. Therefore, influences of a polymerase variant, whichmay be created by substitution of amino acid residues of HBsAg withalanines, upon HBV DNA replication, were assayed. The assayed resultsrevealed that no HBV DNA replication occurs if 163W and 164E are allmutated. In particular, as a result of studying HBV DNA replication whenboth the 163W and 164E were respectively substituted with alanine, the164E variant had HBV DNA replication of about 30 to 70% while the 163Wvariant showed no replication. Therefore, it was identified that aminoacid sites in the polymerase corresponding to 163W site are veryimportant for replication.

164E variants with HBsAg expression and HBV DNA replication were assayedto identify HBV neutralization ability of the inventive antibody. Fromresults thereof, it was confirmed that the HBV neutralization ability isconsiderably decreased because the inventive antibody has a bindingability reduced to about 70%, compared to the wild type. However, forthe 164D variant as a natural variant known in the art, the inventiveantibody exhibited similar binding ability as the wild type.

TABLE 5 Neutralization efficacy of inventive antibody in relation toHBsAg mutation and influence thereof upon HBV DNA replication HBsAgPolymerase Inventive HBV Neutral- mutation mutation antibody Genedia DNAization Mutant Before After Before After plate plate replicationefficacy M5-1 WE AA MGV SRL − Binding − ND M5-2 AA SRV − Binding − NDM5-3 AA SGL − Binding − ND M5-4 AA SGV − Binding − ND M5-5 AA SRV +++Binding − ND M5-6 AE SGV ++ Binding − ND M5-7 WA MGL + Binding ++ NoneM5-8 WA MGM + Binding + None M5-9 WA MGV + Binding ++ None M6-1 WD MGL+++ Binding +++ Yes (*) Compared to the wild type, +++: 70 to 100%; ++:30 to 70%; +: 10 to 30%; and −: <1% ND: Verification test ofneutralization ability was not implemented (Not Determined)

As described in the foregoing description, epitopes of the inventiveantibody in HBsAg include 160K (ayw) or 160R(adr), 163W and 164E. Moreparticularly, the site 164E was identified as the most influentialposition for binding the inventive antibody, through experiments usingalanine substitution variants. At present, this position is known to bemutated into 164D and the inventive antibody also showed neutralizationability to the 164D variant. On the other hand, although the site 163Wdoes not significantly participate in binding of the inventive antibody,mutation at this site causes mutation of the polymerase sequence thatimportantly serves to replicate, which in turn influences HBV DNAreplication. Therefore, it may be predicted that the foregoing site is ahighly conservative position, that is, a position at which mutationoccurs very little. In fact, any mutation at 163W has not yet beenreported. Lastly, 160K (for ayw subtype) or 160R (for adr subtype) areamino acid sites to determine serotypes. From results of functionalassay, these were identified to be in close association with HBVreplication, thus being predicted as highly conservative positions atwhich mutation occurs very little.

1. A polynucleotide encoding a hepatitis B virus (HBV) specific epitope,said epitope comprising RFLWE (SEQ ID NO: 4) or KFLWE (SEQ ID NO: 5). 2.The polynucleotide according to claim 1, wherein the HBV specificepitope comprises FARFLWEWASVRFSW (SEQ ID NO: 6) or FGKFLWEWASARFSW (SEQID NO: 7).
 3. A recombinant vector including the polynucleotide ofclaim
 1. 4. A recombinant vector including the polynucleotide of claim2.
 5. The recombinant vector of claim 3, further comprising a sequenceencoding a promoter or signal protein which derives expression of theHBV specific epitope on the surface of a microorganism cell or virus, ormammalian cells.
 6. The recombinant vector of claim 4, furthercomprising a sequence encoding a promoter or signal protein which allowsan expression of the HBV specific epitope on the surface of amicroorganism cell or virus, or mammalian cells.
 7. A host carrying therecombinant vector of claim 3, wherein said host is a recombinantmicroorganism, virus, or a mammalian cell transformed by the recombinantvector of claim
 3. 8. The host of claim 7, wherein the transformedrecombinant microorganism, virus, or mammalian cell is selected from thegroup consisting of recombinant E. coli, recombinant yeasts, recombinantbacteriophages, and recombinant mammalian cells.
 9. A host carrying therecombinant vector of claim 4, wherein said host is a recombinantmicroorganism, virus, or a mammalian cell transformed by the recombinantvector of claim
 4. 10. The host of claim 9, wherein the transformedrecombinant microorganism, virus, or mammalian cell is selected from thegroup consisting of recombinant E. coli, recombinant yeasts, recombinantbacteriophages, and recombinant mammalian cells.
 11. A host carrying therecombinant vector of claim 5, wherein said host is a recombinantmicroorganism, virus, or a mammalian cell transformed by the recombinantvector of claim
 5. 12. The host of claim 11, wherein the transformedrecombinant microorganism, virus, or mammalian cell is selected from thegroup consisting of recombinant E. coli, recombinant yeasts, recombinantbacteriophages, and recombinant mammalian cells.
 13. A host carrying therecombinant vector of claim 6, wherein said host is a recombinantmicroorganism, virus, or a mammalian cell transformed by the recombinantvector of claim
 6. 14. The host of claim 13, wherein the transformedrecombinant microorganism, virus, or mammalian cell is selected from thegroup consisting of recombinant E. coli, recombinant yeasts, recombinantbacteriophages, and recombinant mammalian cells.
 15. A method forpreparing the hepatitis B virus (HBV) specific epitope, comprising:culturing a vector carrying a polynucleotide encoding the HBV specificepitope or a host carrying the vector in a culture medium to express theHBV specific epitope; and recovering the HBV specific epitope from theculture, wherein the HBV specific epitope has the sequence of SEQ ID NO:4, 5, 6, or 7, and wherein the host is a recombinant microorganism,virus, or mammalian cell transformed with the vector.
 16. The method ofclaim 15, wherein the vector further comprises a sequence encoding apromoter or signal protein which allows an expression of the HBVspecific epitope on the surface of the host.
 17. The method of claim 15,wherein the host is selected from the group consisting of recombinant E.coli, recombinant yeasts, recombinant bacteriophages, and recombinantmammalian cells.